title: Nrf2 degron-fused reporter system: a new tool for specific evaluation of Nrf2 inducers.
abstract: The transcription factor Nrf2 is degraded through the proteasome pathway, but is stabilized in response to oxidative and electrophilic stresses, and activates cytoprotective enzyme genes through binding to the antioxidant/electrophile response element (ARE/EpRE). Nrf2 inducers thus have considerable potential as therapeutic drugs. Although ARE-driven reporters are commonly employed to validate Nrf2 inducers, these reporters are relatively nonspecific. We have generated a new reporter, Nrf2d-LacZ, which may prove to be a better tool for validation of Nrf2 inducers. We made the Nrf2d-LacZ reporter by fusing the N-terminus of Nrf2 harboring a Neh2 degron to β-galactosidase (LacZ), and compared its activity in immortalized mouse embryo fibroblasts (MEFs) with conventional ARE-luciferase (ARE-Luc) reporters in 293T cells, and in MEFs. Nrf2d-LacZ was degraded in unstressed conditions, but stabilized upon exposure to stresses. LacZ activity was induced by electrophiles in a dose-dependent manner, and the induction was detected much more rapidly compared with ARE-Luc. Nrf2d-LacZ was activated not only by electrophiles but also by a variety of other Nrf2 inducing stresses. Although ARE-Luc was activated by 12-O-Tetradecanoylphorbol 13-acetate in an Nrf2-independent manner, Nrf2d-LacZ was not activated by TPA, thus emphasizing the specificity of the Nrf2d-LacZ reporter system for validation of Nrf2 inducers.
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Hirotsu
authors.forename: Fumiki
authors.initials: F
authors.lastname: Katsuoka
authors.forename: Ken
authors.initials: K
authors.lastname: Itoh
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D005347:Fibroblasts / Q000378:metabolism*; D007763:Lac Operon / Q000502:physiology; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D013312:Stress, Physiological; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D015534:Trans-Activators